Numerous investigators in the selenium field have proposed that low molecular weight selenocompounds are responsible for the numerous health benefits attributed to selenium, while an alternative proposal is that selenoproteins are likely the more responsible agents. These health benefits include preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process and having roles in mammalian development, male reproduction and immune function. We were amongst the first to propose several years ago that these health benefits are due largely to the presence of selenium in selenoproteins as the amino acid, selenocysteine (Sec). Therefore, to elucidate the role of selenoproteins in cancer prevention, we are characterizing the function of different selenoproteins in the malignancy process. There are at least two selenoproteins, thioredoxin reductase 1 (TR1) and selenoprotein 15 (Sep15), that have roles in both preventing and promoting cancer. As noted in last years report, we had previously shown that the knockdown of TR1 using RNA interference technology in a lung cancer cell line and in a cancer cell line driven by oncogenic k-ras resulted in several of the malignant phenotypes being reversed more towards those of normal cells suggesting that TR1 deficiency is antitumorigenic. In this past year, we have continued to focus on the molecular basis of TR1s role in cancer development and have examined the role of TR1 in apoptosis in a breast cancer cell line in further detail. As noted previously, the breast cancer cell line was selected since we found that TR1 knockdown in breast cancer cells showed a much higher sensitivity to TNF-alpha induced apoptosis. TR1 has been proposed to control various cellular processes by regulating thioredoxin (Trx). We established Trx1 knockdown cells to examine the involvement of Trx as the major target of TR1s function by comparing responses of TR1 and Trx1 knockdown cells to numerous types of stimuli. These studies showed that both knockdown cells showed similar response patterns to hydrogen peroxide treatment (H2O2), but they responded differently to selenium induced-cytotoxicity. TR1 knockdown cells were much more sensitive to selenium treatment whereas Trx1 knockdown cells showed a similar level of sensitivity as the corresponding control cells. We established a TR1 knockdown and subsequent re-expression strategy for searching for another TR1 binding protein which functions only in the stressed condition. We found several candidate, TR1 binding proteins during cellular stress induced by H2O2, TNF-alpha and selenium and are further characterizing these proteins. The reason for the greater sensitivity of the TR1 deficient cell line to selenium toxicity is poorly understood but this was not due to oxidative stress. We are further examining the enhanced sensitivity of malignant TR1 deficient cells to selenium toxicity since a combination of a TR1 inhibitor and selenium may provide a novel avenue in cancer therapy. We are also examining the up-regulation of TR1 in malignant cells. TR1 is a major antioxidant and redox regulator in mammalian cells and it is indeed over-expressed in many cancer cells. There are four different splicing forms of TR1 that occur in rodents. Three of them (variants II-IV) are driven by the same promoter while the third (variant I) may be controlled by a different promoter. We have identified that the most abundant expression form of TR1 in mice (both normal cells and cancer cells) is variant II. This observation indicates that over-expression of TR1 in cancer cells is initiated by the same promoter as normal cells. Elucidation of the initiation mechanism of TR1 expression in cancer cells will be the key to targeting the up-regulation of TR1 in cancer therapy. We are now analyzing the methylation status of the TR1 promoter region and comparing it in normal and cancer cells. This will elucidate whether DNA methylation may play a role in the regulation of TR1 expression. As reported in last years report, we found that the 15kDa selenoprotein (Sep15) appears to play a role in colorectal cancer. We demonstrated that the targeted down-regulation of Sep15 in the murine colon (adeno)carcinoma cell line, CT26, reversed its characteristics that were typical of a cancer cell to resemble those more like normal cells. Recently, we expanded our studies on the investigation of the role of Sep15 into human colon cancer cell lines of varying malignant statuses, and continue to examine the possible function of this selenoprotein and its role in colon cancer. Furthermore, because we hypothesize TR1 and Sep15 to be the two sides of a double-edged sword in having roles in protecting normal cells from cancer and then, once the malignancy is initiated, both selenoenzymes have roles in the promoting cancer, we have begun studying murine CT26 colon cancer cells in which we successfully knocked down both TR1 and Sep15 using RNAi. We are planning on investigating the interactive effects of these two selenoproteins on malignancy in these and other cancer cells of both mouse and human origin. Of specific interest are cancer cell lines with comparatively strongly increased or decreased TR1 and/or Sep15 protein expression. Thus, we have begun an initial screening of several human cancer cell lines from various tissues using both radio-labeling with 75Se as well as western blotting techniques.